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产品中心 分子酶 分子诊断&siRNA相关药物

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T4 RNA Ligase 1

T4 RNA Ligase,T4 Rnl1

价格 ￥600.00 （ 1-2周）
货号 UA070071
规格
数量
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产品介绍

产品介绍 评论(0)

产品规格

  • 分子别名

    T4 RNA Ligase,T4 Rnl1

  • 表达宿主

    E.coli

  • 分子量

    69kDa (Reducing)

  • 纯度

    ＞95% by SDS-PAGE

  • 活性

    10 U/ul

  • 标签

    His Tag

  • 缓冲体系

    10 mM Tris-HCl、50 mM KCl、1 mM DTT、0.1 mM EDTA、50% Glycerol pH 7.4 @ 25°C

  • 储存条件

    Store at -25 ~ -15℃ for 2 years

  • 文献引用

    [1] England, Thomas E , and O. C. Uhlenbeck . "3|[prime]|-Terminal labelling of RNA with T4 RNA ligase." Nature 275.5680(1978):560-1.
    [2] Tessier, Daniel C. , R. Brousseau , and T. Vernet . "Ligation of single-stranded oligodeoxyribonucleotides by T4 RNA ligase." Analytical Biochemistry 158.1(1986):171-178.

背景介绍

  • T4 RNA Ligase 1 is an ATP-dependent enzyme that catalyzes the formation of phosphodiester bonds between the 5'-P and 3'-OH ends of single-stranded RNA, single-stranded DNA, or single nucleotides, either intermolecular or intramolecular. It is suitable for the connection of single-stranded RNA molecules and the 5' end junction constructed by miRNA libraries in NGS. Labeled with RNA 3 'terminal; Cyclization of oligonucleotides; tRNA modification; In 5'-RACE (Rapid Amplification of cDNA Ends) assay, it is used for oligonucleotides to bind to single-stranded cDNA; and the introduction of unnatural amino acids into proteins.

产品组分

  • Name

    Storage Temperature

    quantity

    concentration

    T4 RNA Ligase 1

    -20 ℃

    100 ul or 500 µl

    10 U/µl

    10*Reaction Buffer

    -20 ℃

    1 ml

    10 X

    Adenosine 5'-Triphosphate (ATP)

    -20 ℃

    1 ml

    10 mM

    PEG8000 (RNase free)

    -20 ℃

    1 ml

    50%


操作步骤

  • 1. Set up a 20 μl reaction as follows:

    Reagent

    Volume

    Final Concentration

    10 * Reaction Buffer

    2 ul

    1 X

    Single-stranded RNA with 5´P and 3´OH ends (200 ng-1 µg)

    1 ul

    10-50 ng/ul

    50% PEG8000

    4 ul

    10%

    1mM ATP

    1 ul

    50 µM

    40 μ/ul Rnase inhibitor

    0.5ul

    1 U/µl

    10 U/µl T4 RNA Ligase 1

    1ul

    0.5 U/µl

    DEPC-treated Water

    Up to 20ul

    -

    2. Incubate at 37°C for 1-2 hours.
    For longer oligos, overnight incubation at 16°C for 16h may improve yield.
    3. Incubate at 65°C for 15min to terminate the reaction.

注意事项

  • Please avoid repeated freeze-thaw cycles.

酶活定义
  • One unit is defined as the amount of enzyme required to convert 1 nanomole of 5´-[32P]rA16 into a phosphatase-resistant form in 30 minutes at 37°C.

  • 生物活性

    • In the experimental design, 133nt single-stranded RNA obtained by transcription in vitro was used. The 5 'end triphosphate group of RNA was removed and digested by RppH enzyme to produce 5' monophosphate group RNA, and then the RNA was connected by T4 RNA Ligase 1. This product was added to the 20µl reaction system, incubated at 37℃ for 120min for reaction, and incubated at 65℃ for 15min to inactivate the enzyme. Remove 9μl, add 1μl 10X RNA Loading Buffer, then perform 20% non-denatured polyacrylamide gel electrophoresis, and then stain with Gelred at room temperature for 20 min, and observe the experimental results under UV lamp. As shown in the figure, this product has the following effects.
      Lane 1 Negative Control-1 (negative control with no added enzyme only);
      Lane 2 Negative Control-2 (only negative controls without T4 RNA Ligase 1);
      Lane 3 UA-T4 RNA Ligase 1 2U;
      Lane 4 UA-T4 RNA Ligase 1 5U;
      Lane 5 UA-T4 RNA Ligase 1 10U;
      Lane 5 UA-T4 RNA Ligase 1 20U;

  • 电泳

    • 2μg (R: reducing condition, N: non-reducing condition).

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